Genome-wide CRISPR screen in a mouse model of tumor growth and metastasis.

Genetic screens are powerful tools for identifying genes responsible for diverse phenotypes. Here we describe a genome-wide CRISPR/Cas9-mediated loss-of-function screen in tumor growth and metastasis. We mutagenized a non-metastatic mouse cancer cell line using a genome-scale library with 67,405 single-guide RNAs (sgRNAs). The mutant cell pool rapidly generates metastases when transplanted into immunocompromised mice. Enriched sgRNAs in lung metastases and late-stage primary tumors were found to target a small set of genes, suggesting that specific loss-of-function mutations drive tumor growth and metastasis. Individual sgRNAs and a small pool of 624 sgRNAs targeting the top-scoring genes from the primary screen dramatically accelerate metastasis. In all of these experiments, the effect of mutations on primary tumor growth positively correlates with the development of metastases. Our study demonstrates Cas9-based screening as a robust method to systematically assay gene phenotypes in cancer evolution in vivo.

Heterozygous deletion of the autism-associated gene CHD8 impairs synaptic function through widespread changes in gene expression and chromatin compaction.

Whole-exome sequencing of autism spectrum disorder (ASD) probands and unaffected family members has identified many genes harboring de novo variants suspected to play a causal role in the disorder. Of these, chromodomain helicase DNA-binding protein 8 (CHD8) is the most recurrently mutated. Despite the prevalence of CHD8 mutations, we have little insight into how CHD8 loss affects genome organization or the functional consequences of these molecular alterations in neurons. Here, we engineered two isogenic human embryonic stem cell lines with CHD8 loss-of-function mutations and characterized differences in differentiated human cortical neurons. We identified hundreds of genes with altered expression, including many involved in neural development and excitatory synaptic transmission. Field recordings and single-cell electrophysiology revealed a 3-fold decrease in firing rates and synaptic activity in CHD8+/- neurons, as well as a similar firing-rate deficit in primary cortical neurons from Chd8+/- mice. These alterations in neuron and synapse function can be reversed by CHD8 overexpression. Moreover, CHD8+/- neurons displayed a large increase in open chromatin across the genome, where the greatest change in compaction was near autism susceptibility candidate 2 (AUTS2), which encodes a transcriptional regulator implicated in ASD. Genes with changes in chromatin accessibility and expression in CHD8+/- neurons have significant overlap with genes mutated in probands for ASD, intellectual disability, and schizophrenia but not with genes mutated in healthy controls or other disease cohorts. Overall, this study characterizes key molecular alterations in genome structure and expression in CHD8+/- neurons and links these changes to impaired neuronal and synaptic function.

A Potential Source of Bias in Group-Level EEG Microstate Analysis.

Microstate analysis is a promising technique for analyzing high-density electroencephalographic data, but there are multiple questions about methodological best practices. Between and within individuals, microstates can differ both in terms of characteristic topographies and temporal dynamics, which leads to analytic challenges as the measurement of microstate dynamics is dependent on assumptions about their topographies. Here we focus on the analysis of group differences, using simulations seeded on real data from healthy control subjects to compare approaches that derive separate sets of maps within subgroups versus a single set of maps applied uniformly to the entire dataset. In the absence of true group differences in either microstate maps or temporal metrics, we found that using separate subgroup maps resulted in substantially inflated type I error rates. On the other hand, when groups truly differed in their microstate maps, analyses based on a single set of maps confounded topographic effects with differences in other derived metrics. We propose an approach to alleviate both classes of bias, based on a paired analysis of all subgroup maps. We illustrate the qualitative and quantitative impact of these issues in real data by comparing waking versus non-rapid eye movement sleep microstates. Overall, our results suggest that even subtle chance differences in microstate topography can have profound effects on derived microstate metrics and that future studies using microstate analysis should take steps to mitigate this large source of error.

Study Protocol: Global Research Initiative on the Neurophysiology of Schizophrenia (GRINS) project.

BACKGROUND: Objective and quantifiable markers are crucial for developing novel therapeutics for mental disorders by 1) stratifying clinically similar patients with different underlying neurobiological deficits and 2) objectively tracking disease trajectory and treatment response. Schizophrenia is often confounded with other psychiatric disorders, especially bipolar disorder, if based on cross-sectional symptoms. Awake and sleep EEG have shown promise in identifying neurophysiological differences as biomarkers for schizophrenia. However, most previous studies, while useful, were conducted in European and American populations, had small sample sizes, and utilized varying analytic methods, limiting comprehensive analyses or generalizability to diverse human populations. Furthermore, the extent to which wake and sleep neurophysiology metrics correlate with each other and with symptom severity or cognitive impairment remains unresolved. Moreover, how these neurophysiological markers compare across psychiatric conditions is not well characterized. The utility of biomarkers in clinical trials and practice would be significantly advanced by well-powered transdiagnostic studies. The Global Research Initiative on the Neurophysiology of Schizophrenia (GRINS) project aims to address these questions through a large, multi-center cohort study involving East Asian populations. To promote transparency and reproducibility, we describe the protocol for the GRINS project. METHODS: The research procedure consists of an initial screening interview followed by three subsequent sessions: an introductory interview, an evaluation visit, and an overnight neurophysiological recording session. Data from multiple domains, including demographic and clinical characteristics, behavioral performance (cognitive tasks, motor sequence tasks), and neurophysiological metrics (both awake and sleep electroencephalography), are collected by research groups specialized in each domain. CONCLUSION: Pilot results from the GRINS project demonstrate the feasibility of this study protocol and highlight the importance of such research, as well as its potential to study a broader range of patients with psychiatric conditions. Through GRINS, we are generating a valuable dataset across multiple domains to identify neurophysiological markers of schizophrenia individually and in combination. By applying this protocol to related mental disorders often confounded with each other, we can gather information that offers insight into the neurophysiological characteristics and underlying mechanisms of these severe conditions, informing objective diagnosis, stratification for clinical research, and ultimately, the development of better-targeted treatment matching in the clinic.

Analysing an allelic series of rare missense variants of CACNA1I in a Swedish schizophrenia cohort.

CACNA1I is implicated in the susceptibility to schizophrenia by large-scale genetic association studies of single nucleotide polymorphisms. However, the channelopathy of CACNA1I in schizophrenia is unknown. CACNA1I encodes CaV3.3, a neuronal voltage-gated calcium channel that underlies a subtype of T-type current that is important for neuronal excitability in the thalamic reticular nucleus and other regions of the brain. Here, we present an extensive functional characterization of 57 naturally occurring rare and common missense variants of CACNA1I derived from a Swedish schizophrenia cohort of more than 10 000 individuals. Our analysis of this allelic series of coding CACNA1I variants revealed that reduced CaV3.3 channel current density was the dominant phenotype associated with rare CACNA1I coding alleles derived from control subjects, whereas rare CACNA1I alleles from schizophrenia patients encoded CaV3.3 channels with altered responses to voltages. CACNA1I variants associated with altered current density primarily impact the ionic channel pore and those associated with altered responses to voltage impact the voltage-sensing domain. CaV3.3 variants associated with altered voltage dependence of the CaV3.3 channel and those associated with peak current density deficits were significantly segregated across affected and unaffected groups (Fisher's exact test, P = 0.034). Our results, together with recent data from the SCHEMA (Schizophrenia Exome Sequencing Meta-Analysis) cohort, suggest that reduced CaV3.3 function may protect against schizophrenia risk in rare cases. We subsequently modelled the effect of the biophysical properties of CaV3.3 channel variants on thalamic reticular nucleus excitability and found that compared with common variants, ultrarare CaV3.3-coding variants derived from control subjects significantly decreased thalamic reticular nucleus excitability (P = 0.011). When all rare variants were analysed, there was a non-significant trend between variants that reduced thalamic reticular nucleus excitability and variants that either had no effect or increased thalamic reticular nucleus excitability across disease status. Taken together, the results of our functional analysis of an allelic series of >50 CACNA1I variants in a schizophrenia cohort reveal that loss of function of CaV3.3 is a molecular phenotype associated with reduced disease risk burden, and our approach may serve as a template strategy for channelopathies in polygenic disorders.

Author Correction: Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

The original version of this paper contained an incorrect primer sequence. In the Methods subsection "Rampage libraries," the text for modification 3 stated that the reverse primer used for library indexing was 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGT-3'. The correct sequence of the oligonucleotide used is 5'-CAAGCAGAAGACGGCATACGAGATXXXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'. This error has been corrected in the PDF and HTML versions of the paper.

Comprehensive comparative analysis of 5'-end RNA-sequencing methods.

Specialized RNA-seq methods are required to identify the 5' ends of transcripts, which are critical for studies of gene regulation, but these methods have not been systematically benchmarked. We directly compared six such methods, including the performance of five methods on a single human cellular RNA sample and a new spike-in RNA assay that helps circumvent challenges resulting from uncertainties in annotation and RNA processing. We found that the 'cap analysis of gene expression' (CAGE) method performed best for mRNA and that most of its unannotated peaks were supported by evidence from other genomic methods. We applied CAGE to eight brain-related samples and determined sample-specific transcription start site (TSS) usage, as well as a transcriptome-wide shift in TSS usage between fetal and adult brain.

Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.

Overexpression of mouse neurogenin ( Neurog) 2 alone or in combination with mouse Neurog2/1 in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) can rapidly produce high-yield excitatory neurons. Here, we report a detailed characterization of human neuronal networks induced by the expression of human NEUROG2 together with human NEUROG2/1 in hESCs using molecular, cellular, and electrophysiological measurements over 60 d after induction. Both excitatory synaptic transmission and network firing activity increased over time. Strikingly, inhibitory synaptic transmission and GABAergic cells were identified from NEUROG2/1 induced neurons (iNs). To illustrate the application of such iNs, we demonstrated that the heterozygous knock out of SCN2A, whose loss-of-function mutation is strongly implicated in autism risk, led to a dramatic reduction in network activity in the NEUROG2/1 iNs. Our findings not only extend our understanding of the NEUROG2/1-induced human neuronal network but also substantiate NEUROG2/1 iNs as an in vitro system for modeling neuronal and functional deficits on a human genetic background.-Lu, C., Shi, X., Allen, A., Baez-Nieto, D., Nikish, A., Sanjana, N. E., Pan, J. Q. Overexpression of NEUROG2 and NEUROG1 in human embryonic stem cells produces a network of excitatory and inhibitory neurons.

Biological concepts in human sodium channel epilepsies and their relevance in clinical practice.

OBJECTIVE: Voltage-gated sodium channels (SCNs) share similar amino acid sequence, structure, and function. Genetic variants in the four human brain-expressed SCN genes SCN1A/2A/3A/8A have been associated with heterogeneous epilepsy phenotypes and neurodevelopmental disorders. To better understand the biology of seizure susceptibility in SCN-related epilepsies, our aim was to determine similarities and differences between sodium channel disorders, allowing us to develop a broader perspective on precision treatment than on an individual gene level alone. METHODS: We analyzed genotype-phenotype correlations in large SCN-patient cohorts and applied variant constraint analysis to identify severe sodium channel disease. We examined temporal patterns of human SCN expression and correlated functional data from in vitro studies with clinical phenotypes across different sodium channel disorders. RESULTS: Comparing 865 epilepsy patients (504 SCN1A, 140 SCN2A, 171 SCN8A, four SCN3A, 46 copy number variation [CNV] cases) and analysis of 114 functional studies allowed us to identify common patterns of presentation. All four epilepsy-associated SCN genes demonstrated significant constraint in both protein truncating and missense variation when compared to other SCN genes. We observed that age at seizure onset is related to SCN gene expression over time. Individuals with gain-of-function SCN2A/3A/8A missense variants or CNV duplications share similar characteristics, most frequently present with early onset epilepsy (<3 months), and demonstrate good response to sodium channel blockers (SCBs). Direct comparison of corresponding SCN variants across different SCN subtypes illustrates that the functional effects of variants in corresponding channel locations are similar; however, their clinical manifestation differs, depending on their role in different types of neurons in which they are expressed. SIGNIFICANCE: Variant function and location within one channel can serve as a surrogate for variant effects across related sodium channels. Taking a broader view on precision treatment suggests that in those patients with a suspected underlying genetic epilepsy presenting with neonatal or early onset seizures (<3 months), SCBs should be considered.

High Prevalence of Hepatic Fibrosis, Measured by Elastography, in a Population-Based Study of Mexican Americans.

BACKGROUND & AIMS: Hepatic fibrosis is a primary risk factor for cirrhosis and hepatocellular carcinoma, which affect a disproportionate number of Hispanics in the United States. We aimed to determine the prevalence of significant fibrosis, measured by point shear-wave elastography (pSWE), and determine characteristics of hepatic fibrosis and simple steatosis in a population-based study of Mexican American Hispanics in south Texas. METHODS: Liver stiffness was measured by pSWE, performed by 2 separate operators, for 406 participants in the Cameron County Hispanic Cohort from 2015 through 2017. Significant fibrosis (F2-F4) was defined as median stiffness > 1.34 m/s. Steatosis was determined by ultrasound. All participants underwent a clinical examination that included a comprehensive laboratory analysis and standardized interview about their medical and social history. We calculated weighted prevalence of fibrosis and determined clinical and demographic associations with significant fibrosis (with or without steatosis) and simple steatosis with no/minimal fibrosis using multinomial logistic regression. RESULTS: Fifty-nine participants were excluded due to unreliable pSWE findings or inconclusive ultrasound results, for a final analysis of 347 participants. The prevalence of significant fibrosis was 13.8%; most of these participants (37/42, 88.1%) had no evidence of viral hepatitis or heavy drinking. Levels of liver enzymes were associated with fibrosis and simple steatosis. Indicators of metabolic health (insulin resistance, triglycerides, and cholesterol) were significantly associated with simple steatosis. Fibrosis, but not simple steatosis, was significantly associated with of antibodies against HCV in plasma (odds ratio, 18.9; P = .0138) and non-significantly associated with reduced platelet count (odds ratio, 0.8 per 50x103/µL; 95% CI, 0.5-1.1). Multivariable analyses, as well as sensitivity analyses removing F4 fibrosis and viral or alcoholic etiologies, confirmed our results. CONCLUSION: We estimated the prevalence of fibrosis in a large population of Mexican American Hispanics using pSWE measurements. We found Mexican American Hispanics to have a higher prevalence of fibrosis compared to European and Asian populations, primarily attributable to metabolic disease.

Transfer of peanut allergy from donor to recipient after liver transplant.

31 years old female with a history of contact dermatitis, eczema, allergic rhinitis, pernicious anemia, alopecia areata and latent tuberculosis was treated concurrently with methotrexate along with isoniazid and pyridoxine. Five months into the therapy she developed acute onset jaundice progressing into fulminant liver failure with altered mentation and worsening liver function tests. Extensive workup including serological and histopathological evaluation revealed drug-induced liver injury as the etiology of her liver failure and she underwent a successful orthotropic liver transplant. On post-transplant follow-up at four months, she was noted to have an allergic reaction consisting of a perioral rash and swelling (without anaphylaxis) after receiving a kiss from her significant other who had just eaten a peanut butter chocolate. She denied any history of allergic reaction to peanuts prior to the transplant. Percutaneous skin testing revealed immediate hypersensitivity to peanut, hazelnut, and pecan believed to be acquired newly post-transplant. Further investigation revealed that the organ donor had a documented history of systemic anaphylaxis from the peanut allergy and a positive peanut-specific IgE level. Also, another parallel solid organ recipient (lung transplant) from the same organ donor experienced a serious anaphylactic reaction after peanut exposure. This is a case of food (peanut) allergy transfer from the donor to the recipient after the liver transplant. This case highlights the importance of incorporating known donor allergies as a part of pre-transplant screening, given the potentially serious consequences from the transfer of allergies to a previously anergic recipient.

The de novo autism spectrum disorder RELN R2290C mutation reduces Reelin secretion and increases protein disulfide isomerase expression.

Despite the recent identification of over 40 missense heterozygous Reelin gene (RELN) mutations in autism spectrum disorder (ASD), none of these has been functionally characterized. Reelin is an integral signaling ligand for proper brain development and post-natal synapse function - properties likely disrupted in ASD patients. We find that the R2290C mutation, which arose de novo in an affected ASD proband, and other analogous mutations in arginine-amino acid-arginine domains reduce protein secretion. Closer analysis of RELN R2290C heterozygous neurospheres reveals up-regulation of Protein Disulfide Isomerase A1, best known as an endoplasmic reticulum-chaperone protein, which has been linked to neuronal pathology. This effect is recapitulated in a heterozygous RELN mouse mutant that is characterized by defective Reelin secretion. These findings suggest that both a deficiency in Reelin signaling and pathologic impairment of Reelin secretion may contribute to ASD risk.

Tracks through the genome to physiological events.

NEW FINDINGS: What is the topic of this review? We discuss tools available to access genome-wide data sets that harbour cell-specific, brain region-specific and tissue-specific information on exon usage for several species, including humans. In this Review, we demonstrate how to access this information in genome databases and its enormous value to physiology. What advances does it highlight? The sheer scale of protein diversity that is possible from complex genes, including those that encode voltage-gated ion channels, is vast. But this choice is critical for a complete understanding of protein function in the most physiologically relevant context. Many proteins of great interest to physiologists and neuroscientists are structurally complex and located in specialized subcellular domains, such as neuronal synapses and transverse tubules of muscle. Genes that encode these critical signalling molecules (receptors, ion channels, transporters, enzymes, cell adhesion molecules, cell-cell interaction proteins and cytoskeletal proteins) are similarly complex. Typically, these genes are large; human Dystrophin (DMD) encodes a cytoskeletal protein of muscle and it is the largest naturally occurring gene at a staggering 2.3 Mb. Large genes contain many non-coding introns and coding exons; human Titin (TTN), which encodes a protein essential for the assembly and functioning of vertebrate striated muscles, has over 350 exons and consequently has an enormous capacity to generate different forms of Titin mRNAs that have unique exon combinations. Functional and pharmacological differences among protein isoforms originating from the same gene may be subtle but nonetheless of critical physiological significance. Standard functional, immunological and pharmacological approaches, so useful for characterizing proteins encoded by different genes, typically fail to discriminate among splice isoforms of individual genes. Tools are now available to access genome-wide data sets that harbour cell-specific, brain region-specific and tissue-specific information on exon usage for several species, including humans. In this Review, we demonstrate how to access this information in genome databases and its enormous value to physiology.

Hepatitis C virus testing perspectives among primary care physicians in four large primary care settings.

BACKGROUND: In 1998, the Centers for Disease Control and Prevention (CDC) published Recommendations for Prevention and Control of Hepatitis C Virus (HCV) Infection and HCV-Related Chronic Disease, recommending HCV testing for populations most likely to be infected with HCV. However, the implementation of risk-based screening has not been widely adopted in health care settings, and 45% to 85% of infected U.S. adults remain unidentified. OBJECTIVES: To develop a better understanding of why CDC's 1998 recommendations have had limited success in identifying persons with HCV infection and provide information about how CDC's 2012 Recommendations for the Identification of Chronic Hepatitis C Virus Infection Among Persons Born During 1945-1965 may be implemented more effectively. DESIGN: Qualitative data were collected and analyzed from a multidisciplinary team as part of the Birth Cohort Evaluation to Advance Screening and Testing for Hepatitis C project. RESPONDENTS: Nineteen providers were asked open-ended questions to identify current perspectives, practices, facilitators, and barriers to HCV screening and testing. Providers were affiliated with Henry Ford Hospital, Mount Sinai Hospital, the University of Alabama, and the University of Texas Health Science Center. RESULTS: Respondents reported the complexity of the 1998 recommendations, and numerous indicated risk factors were major barriers to effective implementation. Other hindrances to hepatitis C testing included physician discomfort in asking questions about socially undesirable behaviors and physician uncertainty about patient insurance coverage. CONCLUSION: Implementation of the CDC's 2012 recommendations could be more successful than the 1998 recommendations due to their relative simplicity; however, effective strategies need to be used for dissemination and implementation for full success.

Hybrid network defense model based on fuzzy evaluation.

With sustained and rapid developments in the field of information technology, the issue of network security has become increasingly prominent. The theme of this study is network data security, with the test subject being a classified and sensitive network laboratory that belongs to the academic network. The analysis is based on the deficiencies and potential risks of the network's existing defense technology, characteristics of cyber attacks, and network security technologies. Subsequently, a distributed network security architecture using the technology of an intrusion prevention system is designed and implemented. In this paper, first, the overall design approach is presented. This design is used as the basis to establish a network defense model, an improvement over the traditional single-technology model that addresses the latter's inadequacies. Next, a distributed network security architecture is implemented, comprising a hybrid firewall, intrusion detection, virtual honeynet projects, and connectivity and interactivity between these three components. Finally, the proposed security system is tested. A statistical analysis of the test results verifies the feasibility and reliability of the proposed architecture. The findings of this study will potentially provide new ideas and stimuli for future designs of network security architecture.

Differential functional consequences of GRIN2A mutations associated with schizophrenia and neurodevelopmental disorders.

Human genetic studies have revealed rare missense and protein-truncating variants in GRIN2A, encoding for the GluN2A subunit of the NMDA receptors, that confer significant risk for schizophrenia (SCZ). Mutations in GRIN2A are also associated with epilepsy and developmental delay/intellectual disability (DD/ID). However, it remains enigmatic how alterations to the same protein can result in diverse clinical phenotypes. Here, we performed functional characterization of human GluN1/GluN2A heteromeric NMDA receptors that contain SCZ-linked GluN2A variants, and compared them to NMDA receptors with GluN2A variants associated with epilepsy or DD/ID. Our findings demonstrate that SCZ-associated GRIN2A variants were predominantly loss-of-function (LoF), whereas epilepsy and DD/ID-associated variants resulted in both gain- and loss-of-function phenotypes. We additionally show that M653I and S809R, LoF GRIN2A variants associated with DD/ID, exert a dominant-negative effect when co-expressed with a wild-type GluN2A, whereas E58Ter and Y698C, SCZ-linked LoF variants, and A727T, an epilepsy-linked LoF variant, do not. These data offer a potential mechanism by which SCZ/epilepsy and DD/ID-linked variants can cause different effects on receptor function and therefore result in divergent pathological outcomes.

TRPA5 encodes a thermosensitive ankyrin ion channel receptor in a triatomine insect.

As ectotherms, insects need heat-sensitive receptors to monitor environmental temperatures and facilitate thermoregulation. We show that TRPA5, a class of ankyrin transient receptor potential (TRP) channels absent in dipteran genomes, may function as insect heat receptors. In the triatomine bug Rhodnius prolixus (order: Hemiptera), a vector of Chagas disease, the channel RpTRPA5B displays a uniquely high thermosensitivity, with biophysical determinants including a large channel activation enthalpy change (72 kcal/mol), a high temperature coefficient (Q10 = 25), and in vitro temperature-induced currents from 53°C to 68°C (T0.5 = 58.6°C), similar to noxious TRPV receptors in mammals. Monomeric and tetrameric ion channel structure predictions show reliable parallels with fruit fly dTRPA1, with structural uniqueness in ankyrin repeat domains, the channel selectivity filter, and potential TRP functional modulator regions. Overall, the finding of a member of TRPA5 as a temperature-activated receptor illustrates the diversity of insect molecular heat detectors.

Multiple-feature extracting modules based leak mining system design.

Over the years, human dependence on the Internet has increased dramatically. A large amount of information is placed on the Internet and retrieved from it daily, which makes web security in terms of online information a major concern. In recent years, the most problematic issues in web security have been e-mail address leakage and SQL injection attacks. There are many possible causes of information leakage, such as inadequate precautions during the programming process, which lead to the leakage of e-mail addresses entered online or insufficient protection of database information, a loophole that enables malicious users to steal online content. In this paper, we implement a crawler mining system that is equipped with SQL injection vulnerability detection, by means of an algorithm developed for the web crawler. In addition, we analyze portal sites of the governments of various countries or regions in order to investigate the information leaking status of each site. Subsequently, we analyze the database structure and content of each site, using the data collected. Thus, we make use of practical verification in order to focus on information security and privacy through black-box testing.

Vulnerability assessment of IPv6 websites to SQL injection and other application level attacks.

Given the proliferation of internet connected devices, IPv6 has been proposed to replace IPv4. Aside from providing a larger address space which can be assigned to internet enabled devices, it has been suggested that the IPv6 protocol offers increased security due to the fact that with the large number of addresses available, standard IP scanning attacks will no longer become feasible. However, given the interest in attacking organizations rather than individual devices, most initial points of entry onto an organization's network and their attendant devices are visible and reachable through web crawling techniques, and, therefore, attacks on the visible application layer may offer ways to compromise the overall network. In this evaluation, we provide a straightforward implementation of a web crawler in conjunction with a benign black box penetration testing system and analyze the ease at which SQL injection attacks can be carried out.